Use of Hemacytometer to Count Cells

1) Dilute cells 1:1 with trypan blue solution

a. Use separate eppendorf tube

b. 100 µL cells, 100 µL trypan blue is a good volume

2) Mix well and allow dye to permeate dead cells (~ 5 min)

3) Place cover on hemacytometer

4) Pipette cell/dye mixture into side channel until one side under cover is filled

5) Count only living cells (not blue) in large central block

6) Multiply by 2 to account for dilution

7) Multiply by 10⁴ to obtain cells/mL

8) Repeat with other side of hemacytometer for average

9) Clean hemacytometer and cover by wiping with ethanol on a Kimwipe

Notes:

1) Total # cells/mL of original cell suspension = (# of cells in 4 boxes/4)x10,000xdf

2) Cell viability = # alive cells/ (# alive cells + # dead cells)